This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. PROJECT #2: STEM CELLS AND DIABETIC CARDIOMYOPATHY The central hypothesis of this project is that diabetes produces negative effects on the growth reserve of cardiac stem cells (CSC) so that the enhanced cell's death cannot be counteracted by repopulating cells that preserve the architecture and function of the myocardium. To test this hypothesis, the murine models of type 1 and 2 diabetes (T1D, T2D) will be used in the following specific aims (SA): 1. SA1 is to examine whether CSC from normal mice will exhibit differences of stemness and pluripotency compared with CSC from diabetic mice. The whole heart of T1D and T2D mice will be used to isolate and identify CSC with c-kit positive, sca1 positive, MDR1 positive, also Lineage negative antigens and/or frozen tissue sections from those tissues will be stained for the markers. The total number of CSC, distribution in cardiac tissue, stem cell proliferation, and in vitro cardiac lineage differentiation will be compared between diabetic and non-diabetic groups. Flow cytometry, immunocytochemistry, immunohistochemistry, and echocardiography techniques will be used in these experiments. 2. SA2 is to investigate how diabetes and hyperglycemia impairs the stemness and cardiac lineage differentiation of CSC isolated from diabetic and non-diabetic hearts. Gene and protein expression patterns in c-kit, sca1, MDR1, AMPK, P53, PI3K/Akt, Nxk2.5, Glut1&4 of CSC from diabetic hearts will be compared with those from non-diabetic CSC. Cardiac lineage markers will be checked before and after in vitro differentiation. To mimic the in vivo diabetic condition, normal CSC of control hearts will also undergo in vitro hyperglycemia treatments. The underlying mechanism related with the genes and protein change under in vivo and in vitro conditions will be explored accordingly. Gene chip, protein array, Western blot, RT-PCR technique, and cell apoptosis/viability/cytotoxicity assays will be used in the analyses. 3. SA3 is to determine whether transplantation of CSC from normal mice will result in greater improvement in cardiac structure and function in infarcted diabetic mouse heart compared with CSC isolated from diabetic mouse heart. The normal mice and diabetic mice will be used to evaluate cardiac function and structural improvements with transplanted CSC. Echocardiography, histological, and pathological techniques will be used in the studies.